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Effects ofα-methyl-p-tyrosine upon cerebral amine metabolism and sleep states in the cat
360
Brain Research, 72 (1974) 360-365 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Effects of a-methyl-p-tyrosine upon cerebral amine metabolism and sleep states in the cat
DONALD STEIN*, MICHEL JOUVET AND JEAN-FRANCOIS PUJOL Department of Experimental Medicine, Claude Bernard University, Lyon (France)
(Accepted February 14th, 1974)
There is recent evidence which can be taken to suggest that an interregulatory relationship may exist between different monoamine systems in the CNS. For example, it has been shown that the inhibition o f catecholamine (CA) synthesis produces an increase of serotonin (5-HT) turnover. This phenomenon has been observed in the rat after intraventricular or intracisternal injection of 6-hydroxydopamine (6-OHDA) 1,7, or after administration of dopamine-fl-oxidase inhibitor 4. Similar increases in serotonin synthesis have been found after destruction of the dorsal norepinephrine bundle in the cat 11,1a. On the basis of this evidence, it seems possible that the biochemical effects of a-methyl-p-tyrosine (AMPT), a potent tyrosine hydroxylase inhibitor 1°,14 would not be restricted only to CA metabolism but would also affect 5-HT metabolism or turnover as well. To test this hypothesis, we decided to study the effects of A M P T administration upon endogenous CA and 5-HT measured in 9 different areas of the cat's brain. The endogenous content of 5-HT, 5-HIAA and tryptophan (TRP) was also determined as an indirect criterion of cerebral 5-HT metabolism. Prior to killing the animals for biochemical analyses, the electrophysiological states of sleep were analyzed for druginduced changes. This was thought necessary because the relationship between A M P T and sleep is still conflicting although there is general agreement concerning the decrease of behavioral and EEG waking z,3,s following A M P T injection, and there are some conflicting reports concerning its effects on paradoxical sleep (PS). Depending upon the laboratory, either decrease 17, no change 3 or increase s has been reported after A M P T administration. The finding of an increase in PS is in disagreement with the hypothesis that this phase of sleep depends upon the activity of CA-containing neurons 6. Nine adult cats weiging approximately 3 kg, of either sex, were used in the neurophysiological study and subsequent biochemical analyses. The animals received chronic implantation of cortical, orbital and muscular electrodes. Following a l-week * Present address: Department of Psychology, Clark University, Worcester, Mass., U.S.A.
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surgical recovery period, 4 cats were injected i.p. at 8 a.m. with 5 ml of 0.9 ~o saline solution. Five animals received an i.p. injection of 200 mg/kg MPT (methyl ester) (Regis Chemical Co.) dissolved in 5 ml of saline. For the biochemical experiment 3 additional cats treated as described above but not recorded were added to each experimental group. Continuous electrophysiological recordings were taken via the electrode implants for an 8-h period. During this time, the cats were housed in a constant temperature incubator (23 zk 1 °C) and behavioral verification of the sleep-waking cycle was made at regular intervals. The analysis of the EEG recordings was done visually for every minute. Minutes of waking, stage 1 sleep (spindles), stage 2 (spindles and/or slow waves) and PS were tabulated during the two consecutive periods of 4 h. At the end of the recording period (4 p.m.), the animals were killed by i.v. injection of saturated KC1 solution and their brains removed. For the biochemical analysis, each brain was rapidly dissected and kept frozen (--20 °C) until assays were performed. Dissection was performed under visual guidance, using classically defined boundaries to separate the tissue. The 9 areas used in the biochemical investigation are listed in Table II. The discrete areas were separately homogenized in ethanol-water (74:26, v/v) and after centrifugation, CA and 5-HT were extracted by passing the solution through an Amberlite C50 column lz. TRP was isolated from the acidified (pH 2) filtrate of the Amberlite column by means of a Dowex column 12 and 5-HIAA was extracted by adsorption upon Sephadex G1012. The concentrations of the different compounds were then determined fluorimetrically as described elsewhere x. (1) Neurophysiological data. The sleep-waking neurophysiological data were separated into two periods for the purpose of analysis: the first period represented the first 4 h after injection, and the second period the second 4-h period. The results of this analysis are presented in Table I. In our hands, there was a significant declease in the
TABLE I RELATIVE DURATION OF SLEEP--WAKINGACTIVITY IN CATS D u r a t i o n o f w a k i n g (W), stage 1 sleep (S1), stage 2 sleep ($2) a n d paradoxical sleep (PS) in salineinjected g r o u p (control, n = 4) a n d A M P T - t r e a t e d cats (200 mg/kg, n = 5). Results are tabulated in percentage o f recorded time (240 min) for the two consecutive periods o f 4 h after the injection.
1-4 h after injection
W SI $2 PS
5-8 h after injection
Control
AMPT
40.5 29.8 26.8 2.5
37.9 29.5 29.6 2.7
± 10.2 4. 3.9 4- 5.6 4- 1
± ± 4. 4-
* P < 0.02 with respect to control. ** P < 0.05 with respect to previous period.
Control 6.6 1.5 4.4 1.6
42 28.3 27.4 5.7
± 44. ±
AMPT 5.7 7.5 7.7 3.7
18.3 34.6 36.9 10
44. ± 4-
5.2* 1.2"* 4.1 2.3**
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TABLE II EFFECTS OF AMPT (200 mg/kg) UPON ENDOGENOUS CA, 5-HT, TRP AND 5-H1AA IN tnFFERENT S T R U C T U R E S OF T H E C A T B R A I N
Cats were killed 8 h after the injection. Results are expressed in percent of mean value measured in a group of saline-treated cats (7 animals) (group 1) ± S.E.M. Statistical differences between AMPTtreated cats (group 2) (8 animals) and saline were estimated by means o f a t test. Numbers in brackets give mean absolute value for each structure.
Structure
NA ( ttg /g )
TRP ( Itg./g )
5-HT ( Itg/g )
5-HIAA ( l'g./g )
Cortex Group 1 Group 2
(0.374) 100 ± 3 64 ± 6**
(7.37) 100 110
(0.293) 100± 7 118_-j~ 3*
(0.277) 100£ 8 1 4 4 ± 9**
Striatum Group 1 Group 2
(0.599) 100 ± 8 61 ~ 5***
(5.55) 100 ± 8 117 -k 18
(0.829) 100±12 109± 7
(0.679) 100~_ 5 96± 7
Thalamus Group 1 Group 2
(0.408) 100 ~F~ 8 68 ± 8*
(7.31 ) 100 } 107 ±
6 7
(0.558) 100 :~ 12 1 1 9 ± 13
(0.986) 100 L 6 110 i:: 6
Hypothalamus Group 1 Group 2
(1.80) 100 _-I 8 91 i 11
(5.72) 100 ~: 5 101 ± 7
(1.28) 100120 78 i 3
(1.08) 1 0 0 ± 14 101 }: 5
Mesencephalon Group 1 Group 2
(0.497) 100 i 7 63 ± 10"**
(6.9) 100 :L 6 129 i 4***
(I.17)
(I.35)
100± 7 115 ~: 5
lOOt: 9 1 3 0 ± 7*
Pons Group 1 Group 2
(0.629) 100 ± 7 65i 6***
(7.87) 100 116"
(0.760) 100± 7 1 2 0 ± 7*
(0.718) 1 0 0 ± 10 145 :~ 6*
Cerebellum Group 1 Group 2
(0.168) 100 4__ 4 30 ± 4***
(9.17) 100 i 6 113 ~ 2*
(0.294) 100 ~ 9 108 ± 10
(0.157) 100 ± 6 154 £ 14"
3 7
DA ( t'g /g )
(12.35) 100±4 46 ± 3 " * *
* P < 0.05. ** P < 0.01. *** P < 0.001.
time the animals were awake after AMPT injection. This change in wakefulness was most clearly seen in the period 4-8 h after treatment (P < 0.02). In general, all phases of sleep increased but no particular stage considered separately was significant with respect to saline-treated controls. (2) Biochemical data. As shown in Table 1I, the administration of AMPT induced a significant decrease of N A content in most but not all of the different brain areas under study: cortex (--36 ~ ) , striatum (--32 ~ ) , thalamus (--32 ~o), mesencephalon (--37 ~ ) , brain stem (--35 ~ ) , and cerebellum (--70 ~ ) . We did not observe any significant change in N A content of the hypothalamus. Striatal dopamine was also significantly decreased by AMPT ( - - 5 4 ~ ) .
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363
The 5-HT concentrations were significantly increased in the cortex (+35~/o) and the brain stem ( + 2 8 ~o). In these two structures the level of 5-HIAA was increased ( + 4 4 ~ and 45 ~ respectively). Significant shifts in 5-HIAA were also observed in the mesencephalon ( + 38 ~o) and cerebellum ( + 54 ~o). In all of the above mentioned structures, T R P concentration was significantly increased. The decrease of cerebral NA and dopamine after A M P T injection in cats can be considered a well established fact and our results are in general agreement with previous findings s,9. We were unable to find any alteration of NA levels in the hypothalamus and in this respect our results support those of King and Jewett 8 and Kitzikis and Roberge 9. Apparently the dynamics o f NA metabolism in the hypothalamus are different from those in other parts o f the brain since Kitzikis and Roberge 9 observed a decrease o f NA only 10 h after A M P T injection. The increase o f 5-HIAA concentrations that we noted in several brain structures is a new finding. This decrease could be interpreted as an index of increasing 5-HT turnover. Indeed, the level o f endogenous 5-HT was either normal or augmented in the areas studied but this increase was always less intense than 5-HIAA elevation. Moreover, the T R P concentration was also increased. This phenomenon has been also reported when drugs which enhance 5-HT synthesis 15,16 are administered. It is noteworthy that the increase o f 5-HIAA usually parallels the decrease of NA. In this study, we have no direct proof of the intimate mechanism by which the inhibition o f NA synthesis induces (directly or indirectly) an increase in 5-HT synthesis. However, our results are in agreement with data reported after other methods o f inhibition of NA synthesis which have been carried out in rats (injection of 6hydroxydopamine 1,7 or dopamine-fl-hydroxylase inhibitor4). Similar results have been obtained after lesions of the dorsal NA pathway in cats TM. Given these results, one might wish to pose the following question: namely, considering the following physiological results, is the alteration of sleep which follows A M P T administration in the cat due only to CA inhibition or might it also be related to increase of 5-HT turnover? Our electrophysiological results confirm the decrease o f waking which has been observed in the cat, and lend support to the hypothesis that some CA neurons may play a role in behavioral and EEG waking 5. After A M P T administration, the decrease o f waking could be obtained through two different and antagonistic mechanisms: on the one hand, the increase o f stage 1 sleep (deactivated EEG) might be due to a passive decrease of activity in the CA neurons involved in EEG arousaP. On the other hand, the increase o f stage 2 sleep might be explained as a consequence of the increase o f turnover in 5-HT neurons 6. The conflicting problem o f the action of A M P T upon PS cannot be solved on the basis o f our data alone. However, our results agree with those o f King and Jewett 8 although we obtained a much smaller increase of PS than in their experiment. In any case, the fact that A M P T does not suppress PS is not in agreement with the hypothesis that CA mechanisms are involved in the triggering of this phase of the sleep cycle. This hypothesis is founded both upon data obtained from lesion studies on suppression of PS after (surgical or 6-OHDA) destruction of the locus coeruleus and subcoeruleus and pharmacological data (selective suppression with a-methyldopa6).
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SHORT COMMUNICATIONS
Since it has been shown that 5-HT mechanisms play a determinant role in the priming o f PS (as shown by the suppression o f PS after inhibition o f 5-HT synthesis 6) the increase o f 5-HT turnover which follows the destruction o f the dorsal N A bundle is accompanied by the parallel increase o f both stage 2 sleep and PS. In some cases, the increase o f PS might be as high as 400 ~o during a period o f 18-24 h after surgery 13. After A M P T administration, the increase o f PS is m u c h smaller, and does not approach significance. It may be that the increase o f 5-HT turnover which normally serves as a priming mechanism for PS cannot fully activate (directly or indirectly) the C A neurons o f the pontine tegmentum since A M P T alters their normal functioning. In summary, the injection o f 200 mg/kg o f DL-AMPT (methyl ester) in cats was followed by a decrease o f waking and increase o f both SWS and PS. As measured in an 8-h period, biochemical analyses performed 8 h after injections showed a decrease o f catecholamines in the brain and an increase o f 5-HT and 5 - H I A A in a number o f different C N S structures. These changes m a y reflect increase o f 5-HT turnover rate. The alteration o f the sleep-waking cycle which followed the injections o f A M P T may be caused by the inhibition o f C A synthesis and c o n c o m i t a n t increase in 5-HT turnover. Support f o r this work by grants f r o m D R M E (72 108), by 1 N S E R M (U 52) and by C N R S (LA 162) are gratefully a c k n o w l e d g e d as is the Fullbright Postdoctoral Fellowship held by D. Stein.
1 BLONDAUX,CH., JUGE, A., SORDET, F., CHOUVET, G., JOUVET, M., ET PUJOL,J. F., Modification du
2 3 4
5 6 7 8 9
10 11 12
m6tabolisme de la s6rotonine (5-HT) c6r6brale induite chez le rat par administration de 6-hydroxydopamine, Brain Research, 50 (1973) 101-114. FUJIMORI,M., AND HIMWlCH,H. E., The effect of alpha methyl P. tyrosine in sleep in rabbits, Fed. Proc., (1971) 541 (Abs.). ISKANDER,T. N., AND KAELBLING,R., Catecholamines, a dream sleep model and depression, Amer. J. Psychiat., 127 (1970) 43-50. JOHNSON, G. A., KIM, E. G., AND BOUKMA, S. S., 5-Hydroxyindole levels in rat brain after inhibition of dopamine-fl-hydroxylase, J. Pharmacol. exp. Ther., 180 (1972) 539-546. JONES,B. E., BOBILLIER,P., PIN, C., ANDJOUVET,M., The effects of lesion of catecholamine containing neurons upon monoamine content of the brain and EEG and behavioural waking in the cat, Brain Research, 58 (1973) 157-177. JOUVET,M., The role of monoamines and acetylcholine containing neurons in the regulation of the sleep waking cycle, Ergebn. Physiol., 64 (1972) 166-307. JUGE, A., SORDET,F., JOUVET,M., ET PUJOL, J. 17, Modification du m6tabolisme de la s6rotonine (5-HT) c6r6brale apr6s 6-hydroxydopamine chez le rat, C.R. Acad. Sci. (Paris), 274 (1972) 3266-3268. KING, D., AND JEWETT, R. E., The effects of a-methyl tyrosine on sleep and brain norepinephrine in cats, J. Pharmacol. exp. Ther., 177 (1971) 188-194. KITSIKIS, A., AND ROBERGE, A. G., Behavioural and biochemical effects of a-methyl tyrosine in cats, Psychopharmacologia (B~rl.), 31 (1973) 143-155. MOORE,K. E., ANDDOMINIC,J'. A., Tyrosine hydroxylase inhibitors, Fed. Proc., 30 (1971) 859-870. PETITJEAN, F., ET JOUVET, M., Hypersomnie et augmentation de l'acide 5-hydroxy-indolac6tique c6r6bral par 16sion isthmique chez le chat, C.R. Sac. Biol. CParis), 164 (1970) 2288-2293. PUJOL,J. F., Contribution d l'Etude des Modifications de la Rdgulation du Mdtabolisme des Monoamines Centralespendant le Sommeil et la Veille, Th6se Doctorat 6s Sciences, Paris, 1970, 192 pp.
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13 PUJOL,J. F., STEIN,D., BLONDAUX,CH., PETITJEAN,F., FROMENT,J. L., ANDJOUVET,M., Biochemical evidences for interaction phenomena between noradrenergic and serotoninergic systems in the cat brain. In E. UsolN (Ed.), Frontiers in Cateeholamines Research, Pergamon Press, New York, 1974, in press. 14 SPECTOR, S., SJOERDSMA, A., AND UDENFRIEND, S., Blockade of endogenous norepinephrine synthesis by a-methyl tyrosine, an inhibitor of tyrosine hydroxylase, J. Pharmacol., 147 (1965) 86-95. 15 TAGLIAMONTE,A., TAGL1AMONTE,P., FORN, J., KRISHNA, G., AND GESSA, F. L., Stimulation of brain serotonin synthesis by dibutyryl cyclic AMP in rats, J. Neurochem., 18 (1971) 1191-1196. 16 TAGLIAMONTE,A., TAGLIAMONTE,P., PEREZ-CRUET,J., AND GESSA,G. L., Increase of brain TRP caused by drugs which stimulate 5-HT synthesis, Nature New Biol., 229 (1971) 125-126. 17 WEITZMAN,E. D., MCGREGOR, P., MOORE, C., AND JACOBY,J., The effect of alpha-methyl-paratyrosine on sleep patterns of the monkey, Life Sci., 8 (1969) 751-758.
Brain Research, 72 (1974) 360-365 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Effects of a-methyl-p-tyrosine upon cerebral amine metabolism and sleep states in the cat
DONALD STEIN*, MICHEL JOUVET AND JEAN-FRANCOIS PUJOL Department of Experimental Medicine, Claude Bernard University, Lyon (France)
(Accepted February 14th, 1974)
There is recent evidence which can be taken to suggest that an interregulatory relationship may exist between different monoamine systems in the CNS. For example, it has been shown that the inhibition o f catecholamine (CA) synthesis produces an increase of serotonin (5-HT) turnover. This phenomenon has been observed in the rat after intraventricular or intracisternal injection of 6-hydroxydopamine (6-OHDA) 1,7, or after administration of dopamine-fl-oxidase inhibitor 4. Similar increases in serotonin synthesis have been found after destruction of the dorsal norepinephrine bundle in the cat 11,1a. On the basis of this evidence, it seems possible that the biochemical effects of a-methyl-p-tyrosine (AMPT), a potent tyrosine hydroxylase inhibitor 1°,14 would not be restricted only to CA metabolism but would also affect 5-HT metabolism or turnover as well. To test this hypothesis, we decided to study the effects of A M P T administration upon endogenous CA and 5-HT measured in 9 different areas of the cat's brain. The endogenous content of 5-HT, 5-HIAA and tryptophan (TRP) was also determined as an indirect criterion of cerebral 5-HT metabolism. Prior to killing the animals for biochemical analyses, the electrophysiological states of sleep were analyzed for druginduced changes. This was thought necessary because the relationship between A M P T and sleep is still conflicting although there is general agreement concerning the decrease of behavioral and EEG waking z,3,s following A M P T injection, and there are some conflicting reports concerning its effects on paradoxical sleep (PS). Depending upon the laboratory, either decrease 17, no change 3 or increase s has been reported after A M P T administration. The finding of an increase in PS is in disagreement with the hypothesis that this phase of sleep depends upon the activity of CA-containing neurons 6. Nine adult cats weiging approximately 3 kg, of either sex, were used in the neurophysiological study and subsequent biochemical analyses. The animals received chronic implantation of cortical, orbital and muscular electrodes. Following a l-week * Present address: Department of Psychology, Clark University, Worcester, Mass., U.S.A.
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SHORT COMMUNICATIONS
surgical recovery period, 4 cats were injected i.p. at 8 a.m. with 5 ml of 0.9 ~o saline solution. Five animals received an i.p. injection of 200 mg/kg MPT (methyl ester) (Regis Chemical Co.) dissolved in 5 ml of saline. For the biochemical experiment 3 additional cats treated as described above but not recorded were added to each experimental group. Continuous electrophysiological recordings were taken via the electrode implants for an 8-h period. During this time, the cats were housed in a constant temperature incubator (23 zk 1 °C) and behavioral verification of the sleep-waking cycle was made at regular intervals. The analysis of the EEG recordings was done visually for every minute. Minutes of waking, stage 1 sleep (spindles), stage 2 (spindles and/or slow waves) and PS were tabulated during the two consecutive periods of 4 h. At the end of the recording period (4 p.m.), the animals were killed by i.v. injection of saturated KC1 solution and their brains removed. For the biochemical analysis, each brain was rapidly dissected and kept frozen (--20 °C) until assays were performed. Dissection was performed under visual guidance, using classically defined boundaries to separate the tissue. The 9 areas used in the biochemical investigation are listed in Table II. The discrete areas were separately homogenized in ethanol-water (74:26, v/v) and after centrifugation, CA and 5-HT were extracted by passing the solution through an Amberlite C50 column lz. TRP was isolated from the acidified (pH 2) filtrate of the Amberlite column by means of a Dowex column 12 and 5-HIAA was extracted by adsorption upon Sephadex G1012. The concentrations of the different compounds were then determined fluorimetrically as described elsewhere x. (1) Neurophysiological data. The sleep-waking neurophysiological data were separated into two periods for the purpose of analysis: the first period represented the first 4 h after injection, and the second period the second 4-h period. The results of this analysis are presented in Table I. In our hands, there was a significant declease in the
TABLE I RELATIVE DURATION OF SLEEP--WAKINGACTIVITY IN CATS D u r a t i o n o f w a k i n g (W), stage 1 sleep (S1), stage 2 sleep ($2) a n d paradoxical sleep (PS) in salineinjected g r o u p (control, n = 4) a n d A M P T - t r e a t e d cats (200 mg/kg, n = 5). Results are tabulated in percentage o f recorded time (240 min) for the two consecutive periods o f 4 h after the injection.
1-4 h after injection
W SI $2 PS
5-8 h after injection
Control
AMPT
40.5 29.8 26.8 2.5
37.9 29.5 29.6 2.7
± 10.2 4. 3.9 4- 5.6 4- 1
± ± 4. 4-
* P < 0.02 with respect to control. ** P < 0.05 with respect to previous period.
Control 6.6 1.5 4.4 1.6
42 28.3 27.4 5.7
± 44. ±
AMPT 5.7 7.5 7.7 3.7
18.3 34.6 36.9 10
44. ± 4-
5.2* 1.2"* 4.1 2.3**
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SHORT COMMUNICATIONS
TABLE II EFFECTS OF AMPT (200 mg/kg) UPON ENDOGENOUS CA, 5-HT, TRP AND 5-H1AA IN tnFFERENT S T R U C T U R E S OF T H E C A T B R A I N
Cats were killed 8 h after the injection. Results are expressed in percent of mean value measured in a group of saline-treated cats (7 animals) (group 1) ± S.E.M. Statistical differences between AMPTtreated cats (group 2) (8 animals) and saline were estimated by means o f a t test. Numbers in brackets give mean absolute value for each structure.
Structure
NA ( ttg /g )
TRP ( Itg./g )
5-HT ( Itg/g )
5-HIAA ( l'g./g )
Cortex Group 1 Group 2
(0.374) 100 ± 3 64 ± 6**
(7.37) 100 110
(0.293) 100± 7 118_-j~ 3*
(0.277) 100£ 8 1 4 4 ± 9**
Striatum Group 1 Group 2
(0.599) 100 ± 8 61 ~ 5***
(5.55) 100 ± 8 117 -k 18
(0.829) 100±12 109± 7
(0.679) 100~_ 5 96± 7
Thalamus Group 1 Group 2
(0.408) 100 ~F~ 8 68 ± 8*
(7.31 ) 100 } 107 ±
6 7
(0.558) 100 :~ 12 1 1 9 ± 13
(0.986) 100 L 6 110 i:: 6
Hypothalamus Group 1 Group 2
(1.80) 100 _-I 8 91 i 11
(5.72) 100 ~: 5 101 ± 7
(1.28) 100120 78 i 3
(1.08) 1 0 0 ± 14 101 }: 5
Mesencephalon Group 1 Group 2
(0.497) 100 i 7 63 ± 10"**
(6.9) 100 :L 6 129 i 4***
(I.17)
(I.35)
100± 7 115 ~: 5
lOOt: 9 1 3 0 ± 7*
Pons Group 1 Group 2
(0.629) 100 ± 7 65i 6***
(7.87) 100 116"
(0.760) 100± 7 1 2 0 ± 7*
(0.718) 1 0 0 ± 10 145 :~ 6*
Cerebellum Group 1 Group 2
(0.168) 100 4__ 4 30 ± 4***
(9.17) 100 i 6 113 ~ 2*
(0.294) 100 ~ 9 108 ± 10
(0.157) 100 ± 6 154 £ 14"
3 7
DA ( t'g /g )
(12.35) 100±4 46 ± 3 " * *
* P < 0.05. ** P < 0.01. *** P < 0.001.
time the animals were awake after AMPT injection. This change in wakefulness was most clearly seen in the period 4-8 h after treatment (P < 0.02). In general, all phases of sleep increased but no particular stage considered separately was significant with respect to saline-treated controls. (2) Biochemical data. As shown in Table 1I, the administration of AMPT induced a significant decrease of N A content in most but not all of the different brain areas under study: cortex (--36 ~ ) , striatum (--32 ~ ) , thalamus (--32 ~o), mesencephalon (--37 ~ ) , brain stem (--35 ~ ) , and cerebellum (--70 ~ ) . We did not observe any significant change in N A content of the hypothalamus. Striatal dopamine was also significantly decreased by AMPT ( - - 5 4 ~ ) .
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363
The 5-HT concentrations were significantly increased in the cortex (+35~/o) and the brain stem ( + 2 8 ~o). In these two structures the level of 5-HIAA was increased ( + 4 4 ~ and 45 ~ respectively). Significant shifts in 5-HIAA were also observed in the mesencephalon ( + 38 ~o) and cerebellum ( + 54 ~o). In all of the above mentioned structures, T R P concentration was significantly increased. The decrease of cerebral NA and dopamine after A M P T injection in cats can be considered a well established fact and our results are in general agreement with previous findings s,9. We were unable to find any alteration of NA levels in the hypothalamus and in this respect our results support those of King and Jewett 8 and Kitzikis and Roberge 9. Apparently the dynamics o f NA metabolism in the hypothalamus are different from those in other parts o f the brain since Kitzikis and Roberge 9 observed a decrease o f NA only 10 h after A M P T injection. The increase o f 5-HIAA concentrations that we noted in several brain structures is a new finding. This decrease could be interpreted as an index of increasing 5-HT turnover. Indeed, the level o f endogenous 5-HT was either normal or augmented in the areas studied but this increase was always less intense than 5-HIAA elevation. Moreover, the T R P concentration was also increased. This phenomenon has been also reported when drugs which enhance 5-HT synthesis 15,16 are administered. It is noteworthy that the increase o f 5-HIAA usually parallels the decrease of NA. In this study, we have no direct proof of the intimate mechanism by which the inhibition o f NA synthesis induces (directly or indirectly) an increase in 5-HT synthesis. However, our results are in agreement with data reported after other methods o f inhibition of NA synthesis which have been carried out in rats (injection of 6hydroxydopamine 1,7 or dopamine-fl-hydroxylase inhibitor4). Similar results have been obtained after lesions of the dorsal NA pathway in cats TM. Given these results, one might wish to pose the following question: namely, considering the following physiological results, is the alteration of sleep which follows A M P T administration in the cat due only to CA inhibition or might it also be related to increase of 5-HT turnover? Our electrophysiological results confirm the decrease o f waking which has been observed in the cat, and lend support to the hypothesis that some CA neurons may play a role in behavioral and EEG waking 5. After A M P T administration, the decrease o f waking could be obtained through two different and antagonistic mechanisms: on the one hand, the increase o f stage 1 sleep (deactivated EEG) might be due to a passive decrease of activity in the CA neurons involved in EEG arousaP. On the other hand, the increase o f stage 2 sleep might be explained as a consequence of the increase o f turnover in 5-HT neurons 6. The conflicting problem o f the action of A M P T upon PS cannot be solved on the basis o f our data alone. However, our results agree with those o f King and Jewett 8 although we obtained a much smaller increase of PS than in their experiment. In any case, the fact that A M P T does not suppress PS is not in agreement with the hypothesis that CA mechanisms are involved in the triggering of this phase of the sleep cycle. This hypothesis is founded both upon data obtained from lesion studies on suppression of PS after (surgical or 6-OHDA) destruction of the locus coeruleus and subcoeruleus and pharmacological data (selective suppression with a-methyldopa6).
364
SHORT COMMUNICATIONS
Since it has been shown that 5-HT mechanisms play a determinant role in the priming o f PS (as shown by the suppression o f PS after inhibition o f 5-HT synthesis 6) the increase o f 5-HT turnover which follows the destruction o f the dorsal N A bundle is accompanied by the parallel increase o f both stage 2 sleep and PS. In some cases, the increase o f PS might be as high as 400 ~o during a period o f 18-24 h after surgery 13. After A M P T administration, the increase o f PS is m u c h smaller, and does not approach significance. It may be that the increase o f 5-HT turnover which normally serves as a priming mechanism for PS cannot fully activate (directly or indirectly) the C A neurons o f the pontine tegmentum since A M P T alters their normal functioning. In summary, the injection o f 200 mg/kg o f DL-AMPT (methyl ester) in cats was followed by a decrease o f waking and increase o f both SWS and PS. As measured in an 8-h period, biochemical analyses performed 8 h after injections showed a decrease o f catecholamines in the brain and an increase o f 5-HT and 5 - H I A A in a number o f different C N S structures. These changes m a y reflect increase o f 5-HT turnover rate. The alteration o f the sleep-waking cycle which followed the injections o f A M P T may be caused by the inhibition o f C A synthesis and c o n c o m i t a n t increase in 5-HT turnover. Support f o r this work by grants f r o m D R M E (72 108), by 1 N S E R M (U 52) and by C N R S (LA 162) are gratefully a c k n o w l e d g e d as is the Fullbright Postdoctoral Fellowship held by D. Stein.
1 BLONDAUX,CH., JUGE, A., SORDET, F., CHOUVET, G., JOUVET, M., ET PUJOL,J. F., Modification du
2 3 4
5 6 7 8 9
10 11 12
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